Hybridoma clones, monoclonal antibodies to VSIG-4, and methods of making and using

ABSTRACT

Described herein are monoclonal antibodies that bind to VSIG-4 including antibodies that block the binding of VSIG-4 to SIGLEC-7, compositions including the monoclonal antibodies, and methods of making and using those antibodies and compositions.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 62/524,821, filed Jun. 26, 2017, which is incorporated by referenceherein.

BACKGROUND

V-set and Ig domain-containing 4 (VSIG-4 or VSIG4) is a C3 complementreceptor, a B7 family-related protein, and a negative regulator of Tcell activation. VSIG-4 expression has been observed to be restricted totissue macrophages, and it has been shown to be down-regulated inresponse to lipopolysaccharide (LPS) (Vogt et al. (2006) J. of Clin.Invest. 116:2817). In healthy tissue, VSIG-4 is expressed ontissue-resident macrophages and functions as a complement receptor toopsonize bacterial pathogens. Massive infiltrates of VSIG-4⁺ macrophagesinto the tumor microenvironment have been observed in patients diagnosedwith non-small cell lung cancer (Liao et al. (2014) Lab. Invest.94:706), and high VSIG-4 expression has also been correlated withhigh-grade glioma and poor patient prognosis (Xu et al. (2015) Am. J.Transl. Res. 7:1172).

SUMMARY OF THE INVENTION

In some aspects, this disclosure describes an antibody that binds toVSIG-4. In some embodiments, the antibody abrogates binding of VSIG-4 toa VSIG-4 ligand. In some embodiments, the antibody abrogates the bindingof VSIG-4 to Sialic acid-binding Ig-like lectin 7 (SIGLEC-7, SIGLEC7,Siglec-7, or Siglec7).

In some embodiments, the antibody may be produced by at least one of thefollowing clones deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110, USA, on May 11,2017: Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms×hVSIG4528903.111 (ATCC Accession No. PTA-124188); Ms×hVSIG4 528905.11 (ATCCAccession No. PTA-124189); Ms×hVSIG4 528906.11 (ATCC Accession No.PTA-124178); Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180); Ms×hVSIG4528912.11 (ATCC Accession No. PTA-124181); Ms×hVSIG4 528922.111 (ATCCAccession No. PTA-124182); Ms×hVSIG4 528927.111 (ATCC Accession No.PTA-124183); Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and Rt×hVSIG4489518.11 (ATCC Accession No. PTA-124186). These deposits were made inaccordance with the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure.

In some embodiments, the antibody includes at least one of a heavy chainvariable region and a light chain variable region of a monoclonalantibody produced by at least one of the following clones: Ms×hVSIG4528902.11 (ATCC Accession No. PTA-124187); Ms×hVSIG4 528903.111 (ATCCAccession No. PTA-124188); Ms×hVSIG4 528905.11 (ATCC Accession No.PTA-124189); Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms×hVSIG4528910.111 (ATCC Accession No. PTA-124180); Ms×hVSIG4 528912.11 (ATCCAccession No. PTA-124181); Ms×hVSIG4 528922.111 (ATCC Accession No.PTA-124182); Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184); Rt×hVSIG4489517.111 (ATCC Accession No. PTA-124185); and Rt×hVSIG4 489518.11(ATCC Accession No. PTA-124186).

In some embodiments, the antibody includes at least one of a heavy chainvariable region comprising the complementarity determining regions(CDRs) of the heavy chain variable region of a monoclonal antibodyproduced by at least one of the following clones and a light chainvariable region comprising the complementarity determining regions(CDRs) of the light chain variable region of a monoclonal antibodyproduced by at least one of the following clones: Ms×hVSIG4 528902.11(ATCC Accession No. PTA-124187); Ms×hVSIG4 528903.111 (ATCC AccessionNo. PTA-124188); Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms×hVSIG4 528908.11(ATCC Accession No. PTA-124179); Ms×hVSIG4 528910.111 (ATCC AccessionNo. PTA-124180); Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms×hVSIG4528927.111 (ATCC Accession No. PTA-124183); Rt×hVSIG4 489509.11 (ATCCAccession No. PTA-124184); Rt×hVSIG4 489517.111 (ATCC Accession No.PTA-124185); and Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

In some embodiments, the antibody may be coupled directly or indirectlyto a detectable marker. In some embodiments, the antibody includes anIgG antibody.

In some embodiments, the antibody includes an antigen-binding fragmentincluding, for example, a Fab fragment, a Fab′ fragment, a F(ab)₂fragment, and a Fv fragment.

In some embodiments, the antibody may abrogate binding of VSIG-4 to aVSIG-4 ligand. In some embodiments, a VSIG-4 ligand can includeSIGLEC-7. In some embodiments, at least one of VSIG-4 and the VSIG-4ligand may be on a cell surface. In some embodiments, VSIG-4 may beexpressed on the surface of a macrophage including, for example, an M2cmacrophage. In some embodiments, the antibody may abrogate a cell-cellinteraction.

In some embodiments, the antibody may bind to an extracellular domain ofVSIG-4. In some embodiments, the antibody may bind to a VSIG-4polypeptide. In some embodiments, the antibody may bind to bothglycosylated and unglycosylated VSIG-4.

This disclosure further describes compositions and kits including anantibody described herein.

In another aspect, this disclosure describes a method of treating amammalian cancer that includes exposing a mammal including a mammaliancancer cell to an antibody described herein.

In some embodiments, VSIG-4 expression is amplified in a patient samplecomprising the mammalian cancer cell. In some embodiments, VSIG-4expression is amplified in a macrophage of the patient sample comprisingthe mammalian cancer cell. In some embodiments, the mammalian cancercell comprises a lung cancer cell or a glioma.

In some embodiments, the mammalian cancer cell is also exposed tochemotherapy or radiation therapy.

In a further aspect, this disclosure describes a method of detectingcancer in a mammal including exposing a mammalian cancer cell to anantibody described herein.

In yet another aspect, this disclosure describes a method of treating anautoimmune disease in a mammal that includes exposing a cell of themammal to an antibody described herein.

In an additional aspect, this disclosure is directed to the followinghybridoma cell lines and monoclonal antibodies produced by the followinghybridoma cell lines:

Ms×hVSIG4 528902.11, deposited as ATCC accession number PTA-124187;

Ms×hVSIG4 528903.111, deposited as ATCC accession number PTA-124188;

Ms×hVSIG4 528905.11, deposited as ATCC accession number PTA-124189;

Ms×hVSIG4 528906.11, deposited as ATCC accession number PTA-124178;

Ms×hVSIG4 528908.11, deposited as ATCC accession number PTA-124179;

Ms×hVSIG4 528910.111, deposited as ATCC accession number PTA-124180;

Ms×hVSIG4 528912.11, deposited as ATCC accession number PTA-124181;

Ms×hVSIG4 528922.111, deposited as ATCC accession number PTA-124182;

Ms×hVSIG4 528927.111, deposited as ATCC accession number PTA-124183;

Rt×hVSIG4 489509.11, deposited as ATCC accession number PTA-124184;

Rt×hVSIG4 489517.111, deposited as ATCC accession number PTA-124185; and

Rt×hVSIG4 489518.11, deposited as ATCC accession number PTA-124186.

The term “antibody” as used herein refers to a molecule that contains atleast one antigen binding site that immunospecifically binds to aparticular antigen target of interest. The term “antibody” thus includesbut is not limited to a full length antibody and/or its variants, afragment thereof, peptibodies and variants thereof, monoclonalantibodies (including full-length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (for example, bispecificantibodies) formed from at least two intact antibodies, humanantibodies, humanized antibodies, and antibody mimetics that mimic thestructure and/or function of an antibody or a specified fragment orportion thereof, including single chain antibodies and fragmentsthereof. Binding of an antibody to a target can cause a variety ofeffects, such as but not limited to where such binding modulates,decreases, increases, antagonizes, agonizes, mitigates, alleviates,blocks, inhibits, abrogates and/or interferes with at least one targetactivity or binding, or with receptor activity or binding, in vitro, insitu, and/or in vivo. An antibody of the present disclosure thusencompasses antibody fragments capable of binding to a biologicalmolecule (such as an antigen or receptor) or portions thereof, includingbut not limited to Fab, Fab′ and F(ab′)₂, pFc′, Fd, a single domainantibody (sdAb), a variable fragment (Fv), a single-chain variablefragment (scFv) or a disulfide-linked Fv (sdFv); a diabody or a bivalentdiabody; a linear antibody; a single-chain antibody molecule; and amultispecific antibody formed from antibody fragments. The antibody maybe of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class(for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, thatis, the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies may be synthesized by hybridoma cellsuncontaminated by other immunoglobulin producing cells. Alternatively,the monoclonal antibody may be produced recombinantly including, forexample, by cells stably or transiently transfected with the heavy andlight chain genes encoding the monoclonal antibody.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring engineering of theantibody by any particular method. In some embodiments, the term“monoclonal” is used herein to refers to an antibody that is derivedfrom a clonal population of cells, including any eukaryotic,prokaryotic, or phage clone, and not the method by which the antibodywas engineered.

As used herein, the term “VSIG-4 ligand” refers to a protein bindingpartner of VSIG-4 and can include both a ligand and/or acounter-receptor. An interaction between VSIG-4 and a VSIG-4 may signalin one or both directions.

As used herein, “isolated” refers to material removed from its originalenvironment (for example, the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate.

As used herein, “room temperature” is 16° C. to 26° C. or, morepreferably, 18° C. to 24° C. In some embodiments, “room temperature” is20° C. to 22° C.

As used herein “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, whether anyparticular polypeptide is at least 40 percent (%), at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical toanother polypeptide may be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Advances in Applied Mathematics 2:482-489,to find the best segment of homology between two sequences. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, 95% identical to a referencesequence according to the present invention, the parameters are set suchthat the percentage of identity is calculated over the full length ofthe reference polypeptide sequence and that gaps in homology of up to 5%of the total number of amino acids in the reference sequence areallowed.

“Binding affinity” or “affinity binding” refers to the strength of thesum total of noncovalent interactions between a single binding site of amolecule (for example, an antibody) and its binding partner (forexample, an antigen or antigenic epitope). The affinity of a molecule Xfor its partner Y is represented by the dissociation constant (K_(D)),which can generally be determined by using methods known in the art, forexample, using the BIACORE biosensor, commercially available fromBIACORE (GE Healthcare Worldwide, Chicago, Ill.). In some embodiments,antibodies of the present disclosure may be described in terms of theirbinding affinity for VSIG-4. In some embodiments, antibodies of thepresent disclosure include antibodies that interact with an antigenwherein the dissociation constant (K_(D)) is less than or equal to5×10⁻⁶ M, less than or equal to 1×10⁻⁶ M, less than or equal to 5×10⁻⁷M, less than or equal to 1×10⁻⁷ M, less than or equal to 5×10⁻⁸ M, lessthan or equal to 1×10⁻⁸ M, less than or equal to 5×10⁻⁹ M, less than orequal to 1×10⁻⁹ M, less than or equal to 5×10⁻¹⁰ M, less than or equalto 1×10⁻¹⁰ M, less than or equal to 5×10⁻¹¹ M, less than or equal to1×10⁻¹¹ M, less than or equal to 5×10⁻¹² M, less than or equal to1×10⁻¹² M, less than or equal to 5×10⁻¹³ M, less than or equal to1×10⁻¹³ M, less than or equal to 5×10⁻¹⁴ M, less than or equal to1×10⁻¹⁴ M, less than or equal to 5×10⁻¹⁵ M, or less than or equal to1×10⁻¹⁵ M.

As used herein, the term “subject” includes, but is not limited to,humans and non-human vertebrates. In some embodiments, a subject is amammal, particularly a human. A subject may be an individual. A subjectmay be an “individual,” “patient,” or “host.” Non-human vertebratesinclude livestock animals, companion animals, and laboratory animals.Non-human subjects also include non-human primates as well as rodents,such as, but not limited to, a rat or a mouse. Non-human subjects alsoinclude, without limitation, chickens, horses, cows, pigs, goats, dogs,cats, guinea pigs, hamsters, mink, and rabbits.

As used herein “in vitro” is in cell culture and “in vivo” is within thebody of a subject. As used herein, “isolated” refers to material thathas been either removed from its natural environment (for example, thenatural environment if it is naturally occurring), produced usingrecombinant techniques, or chemically or enzymatically synthesized, andthus is altered “by the hand of man” from its natural state.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (for example, 1 to 5 includes 1,1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows cell-based detection of a Siglec-7/VSIG-4 interaction.Human VSIG-4 (hVSIG4)/eGFP HEK transfectants were incubated withrecombinant human Siglec-7 (rhSiglec-7)/Fc protein (2 μg/mL) plusanti-Fc APC, or a negative control (anti-Fc-APC only). The percentageshown in the upper right quadrant indicates the level of interaction.

FIG. 2(A-B) shows surface plasmon resonance measured using BIACORE (GEHealthcare Worldwide, Chicago, Ill.) for confirmation of aVSIG-4-Siglec-7 protein-protein interaction. FIG. 2A. Assay schematic;FIG. 2B. CM5 chips (GE Healthcare Worldwide, Chicago, Ill.) werecovalently coated with Protein A/G/L (Novus Biologicals, Littleton,Colo.). Fc-tagged Siglec7 protein was captured on the Protein A/G/Lchip. The chip was then exposed to varying amounts of soluble his-taggedVSIG-4 recombinant protein to determine the K_(D) for the bindinginteraction between the two proteins. The binding affinity wasdetermined to be 29.7 nM.

FIG. 3 shows recombinant human Siglec-7 (rhSiglec-7)/Fc protein titrateson hVSIG-4 transfectant cells. hVSIG4/eGFP HEK transfectant cells wereincubated with varying amounts of rhSiglec-7/Fc protein (between 500ng/mL and 6 μg/mL) or a negative control (FAB110A). The percentage shownin the upper right quadrant indicates the level of interaction.

FIG. 4 shows recombinant human VSIG-4 (rhVSIG-4)/Fc protein titrates onhSiglec-7 transfectant cells. hSiglec-7/eGFP HEK transfecant cells wereincubated with varying amounts of rhVSIG-4/Fc protein (between 500 ng/mLand 6 μg/mL) or a negative control (FAB110A). The percentage shown inthe upper right quadrant indicates the level of interaction.

FIG. 5 shows four anti-human VSIG-4 antibodies block the interaction ofVSIG-4 with rhSiglec-7/Fc. hVSIG4/eGFP HEK transfectants were incubatedwith four different anti-hVSIG-4 antibodies (produced by clonesMs×hVSIG4 528903.111 (“528903”), Ms×hVSIG4 528906.11 (“528906”),Ms×hVSIG4 528908.11 (“528908”), and Ms×hVSIG4 528912.11 (“528912”)) at2.5 g/mL to test the antibody's capacity to block the interaction withSiglec-7. 4 μg/mL of rhSiglec-7/Fc protein was used. The percentageshown in the upper right quadrant indicates the level of interaction.

FIG. 6(A-B) shows VSIG-4 is expressed on M2c monocytes polarized witheither 30 ng/mL Dexamethasone for 3 days (FIG. 6A) or 20 ng/mL IL-10 for3 days (FIG. 6B). M2c cells were polarized as described in Example 3.Ms×hVSIG4 528903.111 (“528903”), Ms×hVSIG4 528906.11 (“528906”),Ms×hVSIG4 528908.11 (“528908”), Ms×hVSIG4 528912.11 (“528912”), andMs×hVSIG4 528922.111 (“528922”) are mouse anti-human VSIG-4antibody-expressing clones. Histograms shown were gated on CD14⁺ ZombieVioler cells.

FIG. 7 shows detection of Siglec-7/VSIG-4 interaction in primary M2cpolarized cells. Polarized M2c cells were incubated with the indicatedamounts of human Siglec-7 Fc protein (1 μg/mL to 10 μg/mL) or a negativecontrol (FAB110P) for 30 minutes at room temperature. An anti-Fc PEdetection antibody was added for another 20 minutes. The cells were thenwashed, stained with anti-VSIG4 Alexa Fluor 647 and anti-CD14 FITC for30 minutes. The cells were then washed with RDFII staining buffer andanalyzed on a LSRII Fortessa flow cytometer (BD Biosciences, San Jose,Calif.).

FIG. 8A-FIG. 8C show representations of the architecture and structureof VSIG-4. FIG. 8A. VSIG-4 is a member of the immunoglobulinsuperfamily; this type I transmembrane receptor contains a 19 amino acidsignal peptide followed by the extracellular domain (residues 20-283), atransmembrane domain (residues 284-304), and intracellular domain(residues 305-399), where the residue numbers correspond to Uniprotnumber: Q9Y279. The extracellular domain of VSIG-4 is comprised of twotandem Ig domains followed by a ˜60 residue juxtamembrane sequence. FIG.8B. The structure of the N-terminal VSIG-4 Ig domain (PDB ID 2icc) showsan exemplary V-type Ig domain, the domain of VSIG-4 responsible forbinding to C3b/iC3b. FIG. 8C. A structural prediction of the VSIG-4ectodomain shows the tandem Ig domain structure followed by anunstructured ˜60 residue juxtamembrane region.

FIG. 9A-FIG. 9D shows Siglec7 binds to glycosylated VSIG-4. FIG. 9A.Purified recombinant human VSIG-4 ectodomain (rhVSIG4) expressed in NS0cells was subjected to deglycosylation with an enzyme cocktail andseparated by SDS-PAGE and visualized by silver stain. FIG. 9B-FIG. 9D.Recombinant human Siglec7-Fc (rhSiglec7-Fc) was captured on a BiacoreCM5 chip modified with Protein A/G/L at ˜700 RU capture density andtested for binding with fully glycosylated rhVSIG4 (FIG. 9B),deglycosylated VSIG-4 (FIG. 9C), and CD56/NCAM (FIG. 9D) atconcentrations ranging between 0.5 nM and 500 nM.

FIG. 10A-FIG. 10D show Rt×hVSIG4 489517.111 specifically binds theVSIG-4 polypeptide regardless of glycosylation state as demonstrated byBiacore. Blocking antibody (Rt×hVSIG4 489517.111) was captured on aProtein A/G/L chip at ˜350 RU and tested for binding to mammalian NS0cell-derived rhVSIG4 (FIG. 10A), mammalian derived and fullydeglycosylated rhVSIG4 (FIG. 10B), E. coli derived and free ofglycosylation VSIG-4 (FIG. 10C), and CD56/NCAM, a heavily glycosylatedprotein (FIG. 10D). All analyte proteins, rhVSIG4 and CD56/NCAM weretested at concentrations ranging between 25 pM and 250 nM. ThatRt×hVSIG4 489517.111 binds VSIG-4 regardless of VSIG-4's glycosylationstate and not to CD56/NCAM, a heavily glycosylated molecule with asimilar glycoprofile as VSIG4, demonstrates the specificity of theRt×hVSIG4 489517.111 to the VSIG-4 polypeptide and shows that Rt×hVSIG4489517.111 is not anti-glycosylation specific.

FIG. 11A-FIG. 11D show Ms×hVSIG4 528906.11 specifically binds the VSIG-4polypeptide regardless of glycosylation state as demonstrated byBiacore. Ms×hVSIG4 528906.11 was captured on a Protein A/G/L chip at˜350 RU and tested for binding to mammalian NS0 cell-derived rhVSIG4(FIG. 11A), mammalian derived and fully deglycosylated rhVSIG4 (FIG.11B), E. coli derived and free of glycosylation VSIG-4 (FIG. 11C), andCD56/NCAM, a heavily glycosylated protein (FIG. 11D). All analyteproteins, rhVSIG4, and CD56/NCAM were tested at concentrations rangingbetween 25 pM and 250 nM. That Ms×hVSIG4 528906.11 binds VSIG4regardless of VSIG4's glycosylation state and not to CD56/NCAM, aheavily glycosylated molecule with a similar glycoprofile as VSIG4,demonstrates the specificity of Ms×hVSIG4 528906.11 to the VSIG4polypeptide and shows that Ms×hVSIG4 528906.11 is not anti-glycosylationspecific.

FIG. 12A-FIG. 12D show Ms×hVSIG4 528908.11 specifically binds the VSIG4polypeptide regardless of glycosylation state as demonstrated byBiacore. Ms×hVSIG4 528908.11 was captured on a Protein A/G/L chip at˜350 RU and tested for binding to mammalian NS0 cell-derived rhVSIG4(FIG. 12A), mammalian derived and fully deglycosylated rhVSIG4 (FIG.12B), E. coli derived and free of glycosylation VSIG-4 (FIG. 12C), andCD56/NCAM, a heavily glycosylated protein (FIG. 12D). All analyteproteins, rhVSIG4 and CD56/NCAM were tested at concentrations rangingbetween 25 pM and 250 nM. That the antibody binds VSIG4 regardless ofits glycosylation state and not to CD56/NCAM, a heavily glycosylatedmolecule with a similar glycoprofile as VSIG4, demonstrates thespecificity of Ms×hVSIG4 528908.11 to the VSIG4 polypeptide and showsthat Ms×hVSIG4 528908.11 is not anti-glycosylation specific.

FIG. 13A-FIG. 13D show Ms×hVSIG4 528912.11 specifically binds the VSIG4polypeptide regardless of glycosylation state as demonstrated byBiacore. Ms×hVSIG4 528912.11 was captured on a Protein A/G/L chip at˜350 RU and tested for binding to mammalian NS0 cell-derived rhVSIG4(FIG. 13A), mammalian derived and fully deglycosylated rhVSIG4 (FIG.13B), E. coli derived and free of glycosylation VSIG-4 (FIG. 13C), andCD56/NCAM, a heavily glycosylated protein (FIG. 13D). All analyteproteins, rhVSIG4 and CD56/NCAM were tested at concentrations rangingbetween 25 pM and 250 nM. That Ms×hVSIG4 528912.11 binds VSIG4regardless of VSIG4's glycosylation state and not to CD56/NCAM, aheavily glycosylated molecule with a similar glycoprofile as VSIG4,demonstrates the specificity of Ms×hVSIG4 528912.11 to the VSIG4polypeptide and shows that Ms×hVSIG4 528912.11 is not anti-glycosylationspecific.

FIG. 14 shows that recombinant human Siglec-7 binds specifically toHEK293 cells transfected with recombinant human VSIG-4 fused to eGFP ina dose-dependent manner but recombinant human Siglec-7 does not bind tothe same cell type transfected with recombinant human VSIG3-eGFP.

FIG. 15A-FIG. 15E show a representative anti-human VSIG-4 antibodyblocks the interaction of VSIG-4 with recombinant human Siglec-7/Fc onadherent M2c macrophages polarized with Dexamethasone (adherent cells).M2c macrophages were incubated with Ms×hVSIG4 528908.11 at 10 μg/mL, 40μg/mL or 200 μg/mL to test the antibody's capacity to block theinteraction with rhSiglec-7 at 100 μg/mL, 25 μg/mL or 5 μg/mL.Histograms were gated on CD14⁺ CD206⁺ cells. FIG. 15A-FIG. 15E form acomplete FIG. 15 as shown in the index figure.

FIG. 16A-FIG. 16I show representative dot plots of histograms shown inFIG. 15 of Ms×hVSIG4 528908.11 and recombinant human Siglec-7 titration.Data shown are gated on CD14⁺ and CD206⁺ cells. FIG. 16A-FIG. 16I form acomplete FIG. 16 as shown in the index figure.

FIG. 17A-FIG. 17E show a representative anti-human VSIG-4 antibodyblocks the interaction of VSIG-4 with recombinant human Siglec-7/Fc onsuspension M2c macrophages polarized with Dexamethasone (suspensioncells). M2c macrophages were incubated with Ms×hVSIG4 528908.11 at 10μg/mL, 40 μg/mL or 200 μg/mL to test the antibody's capacity to blockthe interaction with rhSiglec-7 at 100 μg/mL, 25 μg/mL or 5 μg/mL.Histograms were gated on CD14⁺ CD206⁺ cells. FIG. 17A-FIG. 17E form acomplete FIG. 17 as shown in the index figure.

FIG. 18A-FIG. 18I show representative dot plots of histograms shown inFIG. 17 of Ms×hVSIG4 528908.11 and recombinant human Siglec-7 titration.Data shown are gated on CD14⁺ and CD206⁺ cells. FIG. 18A-FIG. 18I form acomplete FIG. 18 as shown in the index figure.

FIG. 19A-FIG. 19C show representative data for ELISA results obtainedfrom direct ELISA testing. FIG. 19A shows Ms×hVSIG4 528902.11 recognizesmonomeric E. coli-expressed VSIG-4, but not NS0-expressed, dimerizedVSIG-4. FIG. 19B shows Ms×hVSIG4 528906.11 recognizes glycosylated andnon-glycosylated VSIG-4 protein equally. FIG. 19C shows Ms×hVSIG4528905.11 recognizes glycosylated VSIG-4 protein but not the E.coli-expressed VSIG-4.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes antibodies capable of specifically bindingV-set and Ig domain-containing 4 (VSIG-4 or VSIG4 also known asComplement Receptor Ig (CRIg) and Z391G); antibodies capable of blockinginteractions between VSIG-4 and its ligands including, for example, anewly observed interaction between VSIG-4 and SIGLEC-7; compositionsincluding the antibodies describes herein; and methods of making andusing those antibodies and compositions.

VSIG-4 acts as a negative regulator of T cell activation, likely playinga role in maintaining T cell tolerance. Thus, over-expression of VSIG-4in inflammatory tumor microenvironments may promote tumor tolerance.Therefore, antibodies that identify VSIG-4 have potential diagnosticvalue to, for example, allow the identification of aggressive gradetumors, provide an indication of potential long-term prognosis in ananimal having a tumor, or for use as an aid to guide treatment. Inaddition, a neutralizing antibody that abrogates VSIG-4-inducedinhibition of T cell activation could allow for an increased immuneresponse against tumor cells.

Using a screen for protein-protein interactions, sialic acid-bindingIg-like lectin-7 (Siglec7) was identified as a potential ligand forVSIG-4.

Siglec7 is an immunoglobulin family monomer expressed on the surface ofnatural killer (NK) cells and CD8⁺ T cells (Nicoll et al. (1999) J.Biol. Chem. 274:34). Siglec7 is expressed on a highly functional subsetof NK cells, and ligation of Siglec7 suppressed NK cell-mediatedfunctions, suggesting Siglec7 may act as an inhibitory receptor (Shao etal. (2016) Scand. J. Immunol. 84:182). Furthermore, expression ofSiglec7 ligands in the tumor microenvironment affects NK cell tumorimmunosurveillance (Jandus (2014) J. Clin. Invest. 124:1810).

Sialic acid-binding, Ig-type lectins (SIGLECS or Siglecs) are typicallyactivated by engagement with sialic-acid modified proteins (or lipids),and the specificity of the interaction is determined both by the Siglectarget molecule (including, for example, the precise glycan modificationof a particular target protein, which is often not limited to a singlesite) as well as the Siglec molecule itself (for example, differentSiglecs may bind to different sets of glycan modifications), and hencethe specificity or promiscuity of a Siglec-target interaction can bedifficult to determine.

As further described herein, therapeutic blockade of the VSIG-4/Siglec7interaction with a neutralizing antibody may serve to releaseVSIG4-induced inhibition and to increase an immune response against thetumor cells.

Antibodies

In some embodiments, this disclosure describes an antibody that binds toVSIG-4 (that is, an anti-VSIG-4 antibody). In some embodiments, theantibody binds to human VSIG-4 (hVSIG-4).

In some embodiments, an antibody that binds to VSIG-4 is a monoclonalantibody. In some embodiments, the antibodies that bind to VSIG-4include monoclonal antibodies produced by the hybridoma cell lines (alsoreferred to herein as clones) listed in Table 1 and/or by recombinantmethods.

In some embodiments, the antibody is an isolated antibody. In someembodiments, the antibodies may be isolated or purified by conventionalimmunoglobulin purification procedures, such as protein A- orG-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In some embodiments, an antibody that binds to VSIG-4 recognizes aVSIG-4 polypeptide. In some embodiments, the VSIG-4 polypeptide is humanVSIG-4 (Uniprot number: Q9Y279; Gene ID 11326) or a fragment thereof. Insome embodiments, the VSIG-4 polypeptide is mouse VSIG-4 (Uniprotnumber: F6TUL9) or a fragment thereof. In some embodiments, an antibodythat binds to VSIG-4 recognizes a non-reduced VSIG-4 polypeptide.

In some embodiments, an antibody that binds to VSIG-4 binds toglycosylated and unglycosylated VSIG-4. In some embodiments, an antibodythat binds to VSIG-4 recognizes a VSIG-4 polypeptide and not aglycan-modification of VSIG-4. In some embodiments, an antibody thatbinds to VSIG-4 recognizes a glycan-modification of VSIG-4. In someembodiments, an antibody that binds to VSIG-4 does not bind to anα(2,8)-linked polysialic acid. In some embodiments, an antibody thatbinds to VSIG-4 binds to an α(2,8)-linked polysialic acid.

In some embodiments, an antibody that binds to VSIG-4 preferably bindsto an extracellular domain of VSIG-4. In some embodiments, theextracellular domain of VSIG-4 includes amino acids 20-283 of Uniprotnumber Q9Y279. An extracellular domain of VSIG-4 may include an Igdomain, as shown in some embodiments, in FIG. 8. In some embodiments, anantibody that binds to VSIG-4 may bind to Ig domain 1 of VSIG-4 (aminoacids 21-131 of Q9Y279). In some embodiments, an antibody that binds toVSIG-4 may bind to Ig domain 2 of VSIG-4 (amino acids 143-226 ofQ9Y279).

In some embodiments, an antibody that binds to VSIG-4 may include aderivative of an antibody that is modified or conjugated by the covalentattachment of any type of molecule to the antibody. Such antibodyderivatives include, for example, antibodies that have been modified byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, toxins, or linkage to a cellular ligand or other protein. Anyof numerous chemical modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, and metabolic synthesis of tunicamycin.Additionally, the derivatives may contain one or more non-classicalamino acids.

An antibody that binds to VSIG-4 may be coupled directly or indirectlyto a detectable marker by techniques well known in the art. A detectablemarker is an agent detectable, for example, by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Usefuldetectable markers include, but are not limited to, fluorescent dyes,chemiluminescent compounds, radioisotopes, electron-dense reagents,enzymes, coenzymes, colored particles, biotin, or digoxigenin. Adetectable marker often generates a measurable signal, such asradioactivity, fluorescent light, color, or enzyme activity. Antibodiesconjugated to detectable agents may be used for diagnostic ortherapeutic purposes. Examples of detectable agents include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance may becoupled or conjugated either directly to the antibody or indirectly,through an intermediate such as, for example, a linker known in the art,using techniques known in the art. See, for example, U.S. Pat. No.4,741,900, describing the conjugation of metal ions to antibodies fordiagnostic use. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, andacetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a luminescent materialincludes luminol; examples of bioluminescent materials includeluciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ¹¹³mIn, ¹¹⁵mIn), technetium (⁹⁹Tc,⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh and⁹⁷Ru. Techniques for conjugating such therapeutic moieties to antibodiesare well-known.

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by at least one of the following hybridomacell lines (also referred to herein as clones): Ms×hVSIG4 528902.11(ATCC Accession No. PTA-124187); Ms×hVSIG4 528903.111 (ATCC AccessionNo. PTA-124188); Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms×hVSIG4 528908.11(ATCC Accession No. PTA-124179); Ms×hVSIG4 528910.111 (ATCC AccessionNo. PTA-124180); Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms×hVSIG4528927.111 (ATCC Accession No. PTA-124183); Rt×hVSIG4 489509.11 (ATCCAccession No. PTA-124184); Rt×hVSIG4 489517.111 (ATCC Accession No.PTA-124185); or Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528902.11 (ATCC Accession No.PTA-124187).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528903.111 (ATCC Accession No.PTA-124188).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528905.11 (ATCC Accession No.PTA-124189).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528906.11 (ATCC Accession No.PTA-124178).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528908.11 (ATCC Accession No.PTA-124179).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528910.111 (ATCC Accession No.PTA-124180).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528912.11 (ATCC Accession No.PTA-124181).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528922.111 (ATCC Accession No.PTA-124182).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Ms×hVSIG4 528927.111 (ATCC Accession No.PTA-124183).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Rt×hVSIG4 489509.11 (ATCC Accession No.PTA-124184).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by Rt×hVSIG4 489517.111 (ATCC Accession No.PTA-124185).

In some embodiments, an antibody that binds to VSIG-4 includes amonoclonal antibody produced by or Rt×hVSIG4 489518.11 (ATCC AccessionNo. PTA-124186).

Also included in the present disclosure are monoclonal antibodiesproduced by progeny or derivatives of these hybridoma cell lines,monoclonal antibodies produced by equivalent or similar hybridoma celllines, and/or recombinant derivatives made thereof. In some embodiments,an antibody that binds to VSIG-4 includes a recombinantly derivedmonoclonal antibody including, for example, a rabbit B cell derivedmonoclonal antibody.

An intact antibody molecule has two heavy (H) chain variable regions(abbreviated herein as V_(H)) and two light (L) chain variable regions(abbreviated herein as V_(L)). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termed“complementarity determining regions” (“CDRs”), interspersed withregions that are more conserved, termed “framework regions” (“FRs”). Theextent of the FRs and CDRs has been precisely defined (see, Kabat et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, and Chothia et al., J. Mol. Biol. 1987; 196: 901-917). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having the same heavy chain as a monoclonal antibody producedby at least one of the following hybridoma cell lines: Ms×hVSIG4528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11. In someembodiments, a monoclonal antibody includes a monoclonal antibody havingthe same light chain as a monoclonal antibody produced by at least oneof the following hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody includes a monoclonal antibody having the same heavy chain andthe same light chain as a monoclonal antibody produced by at least oneof the following hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody can contain one, two, three, four, five, six, or more aminoacid substitutions in the heavy and/or the light chains identified abovewherein the amino acid substitutions do not substantially affect bindingof the antibody to VSIG-4.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having the same V_(H) domain as a monoclonal antibody producedby at least one of the following hybridoma cell lines: Ms×hVSIG4528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11. In someembodiments, a monoclonal antibody includes a monoclonal antibody havingthe same V_(L) domain as a monoclonal antibody produced by at least oneof the following hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody includes a monoclonal antibody having the same V_(H) domain andthe same V_(L) domain as a monoclonal antibody produced by at least oneof the following hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody can contain one, two, three, four, five, six, or more aminoacid substitutions in the V_(H) domains and/or the V_(L) domainsidentified above which do not substantially affect binding of theantibody to VSIG-4.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having at least one CDR of the V_(H) domain of a monoclonalantibody produced by at least one of the following hybridoma cell lines:Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11. Insome embodiments, a monoclonal antibody includes a monoclonal antibodyhaving at least two CDRs of the V_(H) domain of a monoclonal antibodyproduced by at least one of the following hybridoma cell lines:Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11. Insome embodiments, a monoclonal antibody includes a monoclonal antibodyhaving at least three CDRs of the V_(H) domain of a monoclonal antibodyproduced by at least one of the following hybridoma cell lines:Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

Additionally or alternatively, in some embodiments, a monoclonalantibody includes a monoclonal antibody having at least one CDR of theV_(L) domain of a monoclonal antibody produced by at least one of thefollowing hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody includes a monoclonal antibody having at least two CDRs of theV_(L) domain of a monoclonal antibody produced by at least one of thefollowing hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11. In some embodiments, a monoclonalantibody includes a monoclonal antibody having at least three CDRs ofthe V_(L) domain of a monoclonal antibody produced by at least one ofthe following hybridoma cell lines: Ms×hVSIG4 528902.11; Ms×hVSIG4528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11; Ms×hVSIG4528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11; Ms×hVSIG4528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody can contain one, two, three,four, five, six, or more amino acid substitutions in one or more CDRsidentified above which do not substantially affect binding of theantibody to VSIG-4.

In some embodiments, a monoclonal antibody can contain one, two, three,four, five, six, or more amino acid substitutions in one or moreframework regions (FRs). In some embodiments, the substitutions orsubstitutions in the framework regions (FRs) do not substantially affectbinding of the antibody to VSIG-4.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having an amino acid sequence at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence of atleast one CDR of a V_(H) domain of a monoclonal antibody produced by atleast one of the following hybridoma cell lines: Ms×hVSIG4 528902.11;Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11;Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11;Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11;Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having an amino acid sequence at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence of atleast two CDRs of a V_(H) domain of a monoclonal antibody produced by atleast one of the following hybridoma cell lines: Ms×hVSIG4 528902.11;Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11;Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11;Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11;Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody includes a monoclonalantibody having an amino acid sequence at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence of atleast three CDRs of a V_(H) domain of a monoclonal antibody produced byat least one of the following hybridoma cell lines: Ms×hVSIG4 528902.11;Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11;Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11;Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11;Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody includes an antibody havingan amino acid sequence at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to at least one CDR of a V_(L) domain of theantibody expressed by at least one of the following hybridoma celllines: Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody includes an antibody havingan amino acid sequence at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to at least two CDRs of a V_(L) domain of theantibody expressed by at least one of the following hybridoma celllines: Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, a monoclonal antibody includes an antibody havingan amino acid sequence at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to at least three CDRs of a V_(L) domain of theantibody expressed by at least one of the following hybridoma celllines: Ms×hVSIG4 528902.11; Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11;Ms×hVSIG4 528906.11; Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111;Ms×hVSIG4 528912.11; Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111;Rt×hVSIG4 489509.11; Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11.

The antibody may be an antibody from any suitable species. In someembodiments, the antibody may be a mouse antibody. In some embodiments,the antibody may be a rat antibody. In some embodiments, the antibodymay be a rabbit antibody.

In some embodiments, the antibody is an IgG antibody. In someembodiments, the antibody may be an antibody or an IgG subclassincluding, for example, IgG1, IgG2, IgG3 or IgG4. In some embodiments,the antibody may be a mouse IgG of one of the following sub-classes:IgG1, IgG2A, IgG2B, IgG2C and IgG3. In some embodiments, the antibodymay be a rat IgG of one of the following sub-classes: IgG1, IgG2A,IgG2B, or IgG2C.

In some embodiments, the antibody may include a kappa light chain. Insome embodiments, the antibody may include a lambda light chain.

In some embodiments, the monoclonal antibody includes an antigen-bindingfragment including an Fab fragment, an Fab′ fragment, an F(ab)₂fragment, and/or an Fv fragment.

A monoclonal antibody may be obtained by any suitable technique. In someembodiments, an antibody that binds to VSIG-4 may be made by recombinantDNA methods, produced by phage display, and/or produced by combinatorialmethods. DNA encoding an antibody that binds to VSIG-4 may be readilyisolated and sequenced using conventional procedures. In someembodiments, a hybridoma cell described herein may serve as a source ofsuch DNA. Once isolated, the DNA may be transfected into a host cell(including, for example, simian COS cells, Chinese hamster ovary (CHO)cells, human embryonic kidney cells (HEK), or myeloma cells that do nototherwise produce immunoglobulin protein) or introduced into a host cellby genome editing (for example, using a CRISPR-Cas system) to obtain thesynthesis of monoclonal antibodies in a recombinant host cells. The DNAencoding an antibody that binds to VSIG-4 may be modified to, forexample, humanize the antibody.

In some embodiments, the antibody may be a humanized antibody. Anantibody that binds to VSIG-4 may be humanized by any suitable method.Techniques for producing humanized monoclonal antibodies may be found,for example, in Jones et al. (1986) Nature 321:522 and Singer et al.(1993) J. Immunol. 150:2844. For example, humanization of the antibodymay include changes to the antibody to reduce the immunogenicity of theantibody when used in humans. In some embodiments, a humanized antibodythat binds to VSIG-4 may include at least a portion of an immunoglobulinconstant region (Fc) of a human immunoglobulin. A humanized antibodythat binds to VSIG-4 may include, in some embodiments, a humanimmunoglobulin (recipient antibody) in which residues from one or morecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from one or more CDRs of a non-human speciesantibody (donor antibody), such as mouse, rat, or rabbit antibody, thatbinds to VSIG-4. In some embodiments, Fv framework residues of a humanimmunoglobulin may be replaced by corresponding non-human residues froman antibody that binds to VSIG-4.

In some embodiments, a monoclonal antibody includes a chimeric antibody,that is, an antibody in which different portions are derived fromdifferent animal species. A chimeric antibody may be obtained by, forexample, splicing the genes from a mouse antibody molecule withappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological specificity. See, forexample, Takeda et al. (1985) Nature 314:544.

In some embodiments, an antibody includes a bispecific or a bifunctionalantibody. A bispecific or bifunctional antibody is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. A bispecific antibody may be produced by a variety ofmethods including fusion of hybridomas or linking of F(ab′) fragments.See, for example, Songsivilai and Lachmann (1990) Clin. Exp. Immunol.79:315; Kostelny et al. (1992) J. Immunol. 148:1547. In addition,bispecific antibodies may be formed as “diabodies” (Holliger et al.(1993) PNAS USA 90:6444) or “Janusins” (Traunecker et al. (1991) EMBO J.10:3655; Traunecker et al. (1992) Int. J. Cancer Suppl. 7:51).

In some embodiments, an antibody is produced by an animal (including,but not limited to, human, mouse, rat, rabbit, hamster, goat, horse,chicken, or turkey), produced by a cell from an animal, chemicallysynthesized, or recombinantly expressed. The antibody may be purified byany method known in the art for purification of an immunoglobulinmolecule, for example, by chromatography (for example, ion exchange,affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, an antibody may be fused to aheterologous polypeptide sequence, as described herein or otherwiseknown in the art, including, for example, to facilitate purification.

A monoclonal antibody may be assayed for immunospecific binding by themethods described herein and by any suitable method known in the art.The immunoassay that may be used includes but is not limited to acompetitive and/or a non-competitive assay system using a technique suchas BIACORE analysis, fluorescence activated cell sorter (FACS) analysis,immunofluorescence, immunocytochemistry, Western blot,radio-immunoassay, enzyme linked immunosorbent assay (ELISA), “sandwich”immunoassay, immunoprecipitation assay, precipitin reaction, geldiffusion precipitin reaction, immunodiffusion assay, agglutinationassay, complement-fixation assay, immunoradiometric assay, fluorescentimmunoassay, and protein A immunoassay. Such assays are routine and wellknown in the art (see for example, Ausubel et al., eds, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., N.Y.(1994)).

In some embodiments, an antibody that binds to VSIG-4 includes anantibody that abrogates binding of VSIG-4 to a VSIG-4 ligand. In someembodiments, the antibody includes an antibody that abrogates binding ofVSIG-4 to Siglec-7. In some embodiments the Siglec-7 is human Siglec-7(Uniprot number: Q9Y286). In some embodiments, the antibody may decreasethe binding of VSIG-4 to a VSIG-4 ligand (including, for example,Siglec-7) by at least 10 percent (%), at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 98%.

In some embodiments, a VSIG-4 ligand may be anchored a cell via atransmembrane domain. In some embodiments, a VSIG-4 ligand may be insolution.

In some embodiments, an antibody that abrogates binding of VSIG-4 toSiglec-7 includes a monoclonal antibody produced by at least one of thefollowing hybridoma cell lines: Ms×hVSIG4 528903.111; Ms×hVSIG4528908.11; Ms×hVSIG4 528912.11; Rt×hVSIG4 489509.11; Rt×hVSIG4489517.111; or Rt×hVSIG4 489518.11.

In some embodiments, at least one of the VSIG-4 and the VSIG-4 ligandmay be on a cell surface. For example, VSIG-4 may be expressed on thesurface of a macrophage including, for example, an M2 macrophage. An M2macrophage may include one or more M2 subtypes including M2a, M2b, M2cand M2d subtypes. In some embodiments, a macrophage may preferablyinclude an M2c macrophage. When at least one of the VSIG-4 and theVSIG-4 ligand are on the surface of a cell, an antibody that binds toVSIG-4 may abrogate binding of VSIG-4 to a cell expressing a VSIG-4ligand, binding of a cell expressing VSIG-4 to a VSIG-4 ligand, and/orbinding of a cell expressing VSIG-4 to a cell expressing a VSIG-4ligand. For example, when VSIG-4 and the VSIG-4 ligand Siglec-7 areexpressed on the surface of a cell, an antibody that binds to VSIG-4 mayabrogate cell-cell binding.

In some embodiments, an antibody that binds to VSIG4 may be made byimmunizing an animal with the extracellular domain of VSIG4. In someembodiments, an antibody that binds to VSIG4 may be made by immunizingan animal with amino acids 1-283 of human VSIG-4 (Uniprot number:Q9Y279; gene ID 11326). In some embodiments, an antibody that binds toVSIG4 may be made by immunizing an animal with a portion of humanVSIG-4. In some embodiments, the animal may be a mammal. For example,the animal may be a rabbit, a mouse, a goat, a sheet, a llama or a rat.In some embodiments, the animal may be a chicken.

In another aspect, this disclosure describes an isolated polynucleotidemolecule. In some embodiments, the isolated polynucleotide moleculeincludes a nucleotide sequence encoding an antibody. In someembodiments, the isolated polynucleotide molecule includes a nucleotidesequence that has at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to nucleotide sequence encoding an antibodydescribed herein. In some embodiments, the isolated polynucleotidemolecule includes polynucleotides that hybridize under high stringencyto a nucleotide sequence encoding an antibody or a complement thereof.As used herein “stringent conditions” refer to the ability of a firstpolynucleotide molecule to hybridize, and remain bound to, a second,filter-bound polynucleotide molecule in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), and 1 mM EDTA at 65° C., followed by washing in0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al. (eds.), Current Protocolsin Molecular Biology, Vol. 1, Green Publishing Associates, Inc., andJohn Wiley & Sons, Inc., N.Y. (1989), at p. 2.10.3). In someembodiments, the isolated polynucleotide molecule includespolynucleotides that encode one or more of the CDRs or the heavy and/orlight chains of a monoclonal antibody of the present invention. Generaltechniques for cloning and sequencing immunoglobulin variable domainsand constant regions are well known. See, for example, Orlandi et al.(1989) Proc. Nat'l Acad. Sci. USA 86:3833.

In another aspect, this disclosure describes recombinant vectorsincluding an isolated polynucleotide of the present invention. Thevector may be, for example, in the form of a plasmid, a viral particle,or a phage. The appropriate DNA sequence may be inserted into a vectorby a variety of procedures. In general, the DNA sequence is insertedinto an appropriate restriction endonuclease site(s) in a vector byprocedures known in the art. Such procedures are deemed to be within thescope of those skilled in the art. Large numbers of suitable vectors andpromoters are known to those of skill in the art, and are commerciallyavailable. The following vectors are provided by way of example.Bacterial vectors include, for example, pQE70, pQE60, pQE-9, pBS, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryoticvectors include, for example, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3,pBPV, pMSG, and pSVL. However, any other plasmid or vector may be used.

In a further aspect, this disclosure also includes a host cellcontaining at least one of the above-described vectors. The host cellmay be a higher eukaryotic cell, such as a mammalian or insect cell, ora lower eukaryotic cell, such as a yeast cell. Or, the host cell may bea prokaryotic cell, such as a bacterial cell, or a plant cell.Introduction of a vector construct into the host cell may be effected byany suitable techniques, such as, for example, calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation.(Davis et al., Basic Methods in Molecular Biology (1986)).

Antibodies of the present invention may be expressed in mammalian cells,yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems may also be employed to producesuch proteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989).

Also included in the present invention are phage display librariesexpressing one or more hypervariable regions from an antibody of thepresent invention, and clones obtained from such a phage displaylibrary. A phage display library is used to produce antibody derivedmolecules. Gene segments encoding the antigen-binding variable domainsof antibodies are fused to genes encoding the coat protein of abacteriophage. Bacteriophage containing such gene fusions are used toinfect bacteria, and the resulting phage particles have coats thatexpress the antibody-fusion protein, with the antigen-binding domaindisplayed on the outside of the bacteriophage. Phage display librariesmay be prepared, for example, using the PH.D.-7 Phage Display PeptideLibrary Kit (Catalog # E8100S) or the PH.D.-12 Phage Display PeptideLibrary Kit (Catalog # E8110S) available from New England Biolabs Inc.,Ipswich, Mass. See, for example, Smith and Petrenko (1997) Chem Rev.97:391-410.

Hybridoma Cell Lines

This disclosure further describes hybridoma cell lines (also referred toherein as “clones”) expressing monoclonal antibodies including, forexample, the following hybridoma cell lines: Ms×hVSIG4 528902.11;Ms×hVSIG4 528903.111; Ms×hVSIG4 528905.11; Ms×hVSIG4 528906.11;Ms×hVSIG4 528908.11; Ms×hVSIG4 528910.111; Ms×hVSIG4 528912.11;Ms×hVSIG4 528922.111; Ms×hVSIG4 528927.111; Rt×hVSIG4 489509.11;Rt×hVSIG4 489517.111; or Rt×hVSIG4 489518.11. In some embodiments, amonoclonal antibody produced by a hybridoma cell line binds to VSIG-4.In some embodiments, a monoclonal antibody produced by a hybridoma cellline abrogates binding of VSIG-4 to Siglec-7.

Hybridoma cell lines may be obtained by various techniques familiar tothose skilled in the art. For example, cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, for example, Kohler and Milstein (1976) Eur. J.Immunol. 6:511; J. Goding in “Monoclonal Antibodies: Principles andPractice,” Academic Press, pp 59-103 (1986); and Harlow et al.,Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub.1988). In some embodiments, the immunized animal is preferably a mammal.In some embodiments, the immunized animal is a rat including, forexample, a Wistar rat, or a mouse including, for example, a BALB/Cmouse. In some embodiments, the cells from the animal are spleen cells.In some embodiments, the cells from the animal are preferablylymphocytes. In some embodiments, the myeloma cell includes aP3X63Ag8.653 cell.

Other known methods of producing transformed B cell lines that producemonoclonal antibodies may also be used.

Recombinant Antibodies

This disclosure further describes recombinantly-derived monoclonalantibodies. Recombinantly derived monoclonal antibodies may include, forexample, rabbit B cell derived monoclonal antibodies. Monoclonalantibodies of the present disclosure may be produced by any suitablerecombinant technique including, for example, by phage display or bycombinatorial methods. See, for example, U.S. Pat. No. 5,223,409; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; or WO 90/02809. Such methods may be used togenerate human monoclonal antibodies.

Uses for the Anti-VSIG-4 Antibodies

An antibody that binds to VSIG-4, as described herein, may be used forany suitable application. For example, a monoclonal antibody may be usedin both in vitro and in vivo diagnostic and therapeutic methods.

In some embodiments, an antibody may be used to determine a level ofexpression of VSIG-4 protein in vitro or in vivo. In some embodiments,determining a level of VSIG-4 protein expression may be used to detectcancer. In some embodiments, an antibody may be used to label a cell invivo or in vitro. In some embodiments, an antibody may be used todetermine a level of expression of VSIG-4 protein in a patient sample.In some embodiments, a patient sample may include a mammalian cancercell. In some embodiments, the mammalian cancer cell may include a lungcancer cell or a glioma cell. In some embodiments, a patient sampleincluding, for example, a patient samples including a mammalian cancercell may include a macrophage.

In some embodiments, the antibody may be labeled. For example, anantibody may be used to label a cell, and the labeled cell may bedirectly or indirectly imaged via secondary methods. In someembodiments, the cell is a mammalian cell.

In some embodiments, an antibody may be used to identify the presence orabsence of VSIG-4 protein in a sample from a subject. In someembodiments, identifying the presence of VSIG-4 may include identifyingan amount of VSIG-4 in a sample from a subject. In some embodiments,identifying the presence of VSIG-4 may be used to detect cancer.

VSIG-4 has previously been reported to be expressed on tissue residentmacrophages. Massive infiltrates of VSIG-4⁺ macrophages into the tumormicroenvironment have been observed in patients diagnosed with non-smallcell lung cancer (Liao et al. (2014) Lab. Invest. 94:706), and highVSIG-4 expression has also been correlated with high-grade glioma andpoor patient prognosis (Xu et al. (2015) Am. J. Transl. Res. 7:1172). Asshown in Example 3, VSIG-4 expression was detected on CD14-positivehuman PBMC monocytes differentiated in vitro to the M2c macrophagelineage, polarized with either Dexamethasone or recombinant human IL-10(rhIL-10) (FIG. 6).

In some embodiments, an anti-VSIG-4 antibody may be used to determinethe level of VSIG-4 expression on a cancer cell, a tumor cell, or apatient sample including a cancer cell or a tumor cell. In someembodiments, a patient sample including a cancer cell or a tumor cellmay further include a macrophage. In some embodiments, an anti-VSIG-4antibody may be used to determine the level of VSIG-4 expression in oron cells of a patient sample including, for example, a macrophage. Insome embodiments, the level of VSIG-4 expression will be amplified. Insome embodiments, an anti-VSIG-4 antibody may be used to determine thelevel of VSIG-4 expression in and/or on a macrophage. In someembodiments, identifying the expression or level of expression of VSIG-4may be used to identify a cancer or a tumor; to assist in the grading ora cancer or a tumor; and/or to provide an indication of long-termprognosis. In some embodiments, the cancer may include a lung cancer ora glioma.

In some embodiments, an anti-VSIG-4 antibody may be used to treat amammalian cancer. In some embodiments, treating a mammalian cancer mayinclude exposing a mammal comprising a mammalian cancer cell ananti-VSIG-4 antibody. In some embodiments, VSIG-4 expression may beamplified in a patient sample that includes the mammalian cancer cell.For example, VSIG-4 expression may be amplified in a macrophage of thepatient sample that includes the mammalian cancer cell. In someembodiments, the mammalian cancer may include a lung cancer or a glioma.

In some embodiments, an anti-VSIG-4 antibody may be used to treat anautoimmune disease. An autoimmune disease may include, for example,autoimmune type 1 diabetes mellitus, rheumatoid arthritis, psoriasis, orlupus. Without wishing to be bound by theory, because macrophages areknown to play a role in the pathogenesis of certain autoimmune diseases,see, e.g., Campos Navegantes et al. (2017) J. Transl. Med. 15:36, and,as shown herein (see Example 5), an anti-VSIG-4 antibody may block theinteraction of naturally expressed VSIG-4 on M2c macrophages withSiglec-7, an anti-VSIG-4 antibody may be used, for example, to treat anautoimmune disease that includes interactions with VSIG-4 expressed onM2c macrophages.

As described in Example 1, soluble, recombinant human VSIG-4 was platedas a capture reagent in a standard ELISA assay. Recombinant,biotinylated human SIGLEC-7 was found to bind to VSIG-4 (FIG. 1 & Table1). The putative protein-protein interaction was then examined in acell-based assay. HEK cells transfected to over-express human VSIG-4 andeGFP were cultured with rhSIGLEC-7/Fc. Binding of rhSIGLEC-7/Fc to thecells was detected with APC-conjugated anti-Fc antibody by flowcytometry (FIG. 2). The binding of SIGLEC-7 to VSIG-4 was furtheranalyzed via Surface Plasmon Resonance (SPR)/BIACORE (FIG. 3).

A cell-based assay was used to titrate the amount of rhSIGLEC-7 onVSIG-4 transfected cells, and the interaction was shown to be dosedependent (FIG. 4). To further confirm this interaction, the assay wasflipped, and rhVSIG-4 was also shown bind SIGLEC-7 expressing cells in adose dependent manner (FIG. 5).

As shown in Examples 2 and 3, the specific binding of rhSIGLEC-7 toVSIG-4 was confirmed using anti-VSIG-4 antibodies to block theinteraction (FIG. 5). Four clones were shown to specifically block theSIGLEC-7/VSIG-4 interaction. The binding affinity of these and othermonoclonal anti-VSIG-4 antibodies were determined via Surface PlasmonResonance (SPR)/BIACORE (Table 2).

As shown in Example 4, VSIG-4 may be glycosylated (FIG. 9). As shown inExample 4 and Example 6, in some embodiments, in some embodiments, ananti-VSIG-4 antibody may bind to the VSIG-4 polypeptide and not to aglycan-modification of VSIG-4 (FIG. 10-FIG. 13, FIG. 19, Table 3),resulting in an anti-VSIG-4 antibody that may bind to both glycosylatedand unglycosylated VSIG-4. In some embodiments an anti-VSIG4 antibodymay bind to the glycosylated VSIG-4, but not the unglycosylatedpolypeptide.

In some embodiments, an anti-VSIG-4 antibody or a composition comprisingan anti-VSIG-4 antibody may be used to abrogate binding of VSIG-4 toSiglec-7. For example, as shown in Example 5, an anti-VSIG-4 antibody ora composition comprising an anti-VSIG-4 antibody may be used to abrogatea cell-cell interaction (FIG. 15-18). Without wishing to be bound bytheory, it is believed that an antibody that abrogates binding of VSIG-4to Siglec-7 could abrogate or inhibit T cell/NK cell immunosurveillanceby ligation of Siglec-7 on CD8⁺ T cells and NK cells. Thus, therapeuticblockade of the VSIG-4-Siglec7 interaction with neutralizing antibodycould serve to release VSIG-4-induced inhibition and to enhance immunefunction against tumor cells or suppress immune responses in anauto-immune disease. Additionally or alternatively, an antibody thatabrogates binding of VSIG-4 to Siglec-7 could abrogate or inhibitmacrophage signaling by inhibiting the binding of Siglec-7 to VSIG-4 ona macrophage.

This disclosure further described a kit including an antibody. Forexample, a kit may include a composition that includes an anti-VSIG-4monoclonal antibody. The antibodies in the kit may be labeled with oneor more detectable markers, as described herein.

A kit may include one or more containers filled with one or more of themonoclonal antibodies of the invention. Additionally, the kit mayinclude other reagents such as buffers and solutions needed to practicethe invention are also included. Optionally associated with suchcontainer(s) may be a notice or printed instructions. As used herein,the phrase “packaging material” refers to one or more physicalstructures used to house the contents of the kit. The packaging materialis constructed by well-known methods, preferably to provide a sterile,contaminant-free environment. As used herein, the term “package” refersto a solid matrix or material such as glass, plastic, paper, foil, andthe like, capable of holding within fixed limits a polypeptide.

Compositions Including Antibodies

In some embodiments, this disclosure describes a composition includingat least one of the antibodies describes herein.

In some embodiments, the composition may also include, for example,buffering agents to help to maintain the pH in an acceptable range orpreservatives to retard microbial growth. A composition may alsoinclude, for example, carriers, excipients, stabilizers, chelators,salts, or antimicrobial agents. Acceptable carriers, excipients,stabilizers, chelators, salts, preservatives, buffering agents, orantimicrobial agents, include, but are not limited to, buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives, such as sodium azide,octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol; polypeptides;proteins, such as serum albumin, gelatin, or non-specificimmunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; aminoacids such as glycine, glutamine, asparagine, histidine, arginine, orlysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (for example, Zinc(Zn)-protein complexes); and/or non-ionic surfactants such as TWEEN,PLURONICS, or polyethylene glycol (PEG).

In some embodiments, the composition is a pharmaceutical composition andincludes the monoclonal antibody and a pharmaceutically acceptablecarrier, diluent or excipient. In the preparation of the pharmaceuticalcompositions comprising the antibodies described in the teachingsherein, a variety of vehicles and excipients may be used, as will beapparent to the skilled artisan.

The pharmaceutical compositions will generally comprise apharmaceutically acceptable carrier and a pharmacologically effectiveamount of an antibody, or mixture of antibodies.

The pharmaceutical composition may be formulated as a powder, a granule,a solution, a suspension, an aerosol, a solid, a pill, a tablet, acapsule, a gel, a topical cream, a suppository, a transdermal patch,and/or another formulation known in the art.

For the purposes described herein, pharmaceutically acceptable salts ofan antibody are intended to include any art-recognized pharmaceuticallyacceptable salts including organic and inorganic acids and/or bases.Examples of salts include but are not limited to sodium, potassium,lithium, ammonium, calcium, as well as primary, secondary, and tertiaryamines, esters of lower hydrocarbons, such as methyl, ethyl, and propyl.Other salts include but are not limited to organic acids, such as aceticacid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, salicylic acid, etc.

As used herein, “pharmaceutically acceptable carrier” comprises anystandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Forexample, the antibody may be prepared as a formulation in apharmaceutically acceptable diluent, including for example, saline,phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose,mannitol, dextran, propylene glycol, oils (for example, vegetable oils,animal oils, synthetic oils, etc.), microcrystalline cellulose,carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesiumstearate, calcium phosphate, gelatin, polysorbate 80 or as a solidformulation in an appropriate excipient.

A pharmaceutical composition will often further comprise one or morebuffers (for example, neutral buffered saline or phosphate bufferedsaline), carbohydrates (for example, glucose, sucrose or dextrans),mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants (for example, ascorbic acid, sodium metabisulfite,butylated hydroxytoluene, butylated hydroxyanisole, etc.),bacteriostats, chelating agents such as EDTA or glutathione, adjuvants(for example, aluminium hydroxide), solutes that render the formulationisotonic, hypotonic or weakly hypertonic with the blood of a recipient,suspending agents, thickening agents and/or preservatives.Alternatively, compositions of the present invention may be formulatedas a lyophilizate.

Any suitable carrier known to those of ordinary skill in the art may beemployed in a composition including at least one of the antibodiesdescribes herein. Antibody compositions may be formulated for anyappropriate manner of administration, including for example, oral,nasal, mucosal, intravenous, intraperitoneal, intradermal, subcutaneous,and intramuscular administration.

Administration and Treatment

The compositions of the present disclosure may be formulated inpharmaceutical preparations in a variety of forms adapted to the chosenroute of administration. One of skill will understand that thecomposition will vary depending on mode of administration and dosageunit. For example, for parenteral administration, isotonic saline may beused. For topical administration a cream, including a carrier such asdimethylsulfoxide (DMSO), or other agents typically found in topicalcreams that do not block or inhibit activity of the peptide, may beused. Other suitable carriers include, but are not limited to alcohol,phosphate buffered saline, and other balanced salt solutions. Thecompounds of this invention may be administered in a variety of ways,including, but not limited to, intravenous, topical, oral, subcutaneous,intraperitoneal, and intramuscular delivery. In some aspects, thecompounds of the present invention may be formulated for controlled orsustained release. In some aspects, a formulation for controlled orsustained release is suitable for subcutaneous implantation. In someaspects, a formulation for controlled or sustained release includes apatch. A compound may be formulated for enteral administration, forexample, formulated as a capsule or tablet.

Administration may be as a single dose or in multiple doses. In someembodiments, the dose is an effective amount as determined by thestandard methods, including, but not limited to, those described herein.Those skilled in the art of clinical trials will be able to optimizedosages of particular compounds through standard studies. Additionally,proper dosages of the compositions may be determined without undueexperimentation using standard dose-response protocols. Administrationincludes, but is not limited to, any of the dosages and dosingschedules, dosing intervals, and/or dosing patterns described in theexamples included herewith.

The composition including an antibody according to the presentdisclosure may be administered by any suitable means including, but notlimited to, for example, oral, rectal, nasal, topical (includingtransdermal, aerosol, buccal and/or sublingual), vaginal, parenteral(including subcutaneous, intramuscular, and/or intravenous),intradermal, intravesical, intra-joint, intra-arteriole,intraventricular, intracranial, intraperitoneal, intranasal, byinhalation, or intralesional (for example, by injection into or around atumor).

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that may be employed will be known tothose of skill in the art. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity, andgeneral safety and purity standards as required by the FDA. Suchpreparations may be pyrogen-free.

Many suitable formulations are known, including polymeric or proteinmicroparticles encapsulating drug to be released, ointments, gels, orsolutions which may be used topically or locally to administer drug, andeven patches, which provide controlled release over a prolonged periodof time. These may also take the form of implants. Such an implant maybe implanted within the tumor.

The compounds of the present invention may also be provided in alyophilized form. Such compositions may include a buffer, for example,bicarbonate, for reconstitution prior to administration, or the buffermay be included in the lyophilized composition for reconstitution with,for example, water. The lyophilized composition may further comprise asuitable vasoconstrictor, for example, epinephrine. The lyophilizedcomposition may be provided in a syringe, optionally packaged incombination with the buffer for reconstitution, such that thereconstituted composition may be immediately administered to a patient.

As used herein “treating” or “treatment” may include therapeutic and/orprophylactic treatments. “Treating a disorder,” as used herein, is notintended to be an absolute term. Treatment may lead to an improvedprognosis or a reduction in the frequency or severity of symptoms. A“therapeutically effective” concentration or amount as used herein is anamount that provides some improvement or benefit to the subject.Desirable effects of treatment include preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, amelioration or palliation of thedisease state, and remission or improved prognosis. Likewise, the term“preventing,” as used herein, is not intended as an absolute term.Instead, prevention refers to delay of onset, reduced frequency ofsymptoms, or reduced severity of symptoms associated with a disorder.Prevention therefore refers to a broad range of prophylactic measuresthat will be understood by those in the art. In some circumstances, thefrequency and severity of symptoms is reduced to non-pathologicallevels. In some circumstances, the symptoms of an individual receivingthe compositions of the invention are only 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10%, 5%, or 1% as frequent or severe as symptoms experiencedby an untreated individual with the disorder.

Therapeutically effective concentrations and amounts may be determinedfor each application herein empirically by testing the compounds inknown in vitro and in vivo systems, such as those described herein,dosages for humans or other animals may then be extrapolated therefrom.

It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions andmethods.

Toxicity and therapeutic efficacy of the compositions may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, for example, for determining the LD₅₀ (the dose lethal to 50%of the population) and the ED₅₀ (the dose therapeutically effective in50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it may be expressed as the ratiobetween LD₅₀ and ED₅₀. Compositions that exhibit high therapeuticindices may be preferred. The data obtained from cell culture assays andanimal studies may be used in formulating a range of dosage for use inhumans. The dosage of such compositions may preferably lie within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage may be chosen by theindividual physician in view of the patient's condition.

A composition as described herein may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. For example, compositions may be administered repeatedly, forexample, at least 2, 3, 4, 5, 6, 7, 8, or more times, or may beadministered by continuous infusion. It is understood that the precisedosage and duration of treatment is a function of the disease beingtreated and may be determined empirically using known testing protocolsor by extrapolation from in vivo or in vitro test data. It is to benoted that concentrations and dosage values may also vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the concentration rangesset forth herein are exemplary only and are not intended to limit thescope or practice of the claimed compositions and methods.

In some therapeutic embodiments, an “effective amount” of an agent is anamount that results in a reduction of at least one pathologicalparameter. Thus, for example, in some aspects of the present disclosure,an effective amount is an amount that is effective to achieve areduction of at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% compared to theexpected reduction in the parameter in an individual not treated withthe agent.

In some aspects of the methods of the present disclosure, a methodfurther includes the administration of one or more additionaltherapeutic agents. One or more additional therapeutic agents may beadministered before, after, and/or coincident to the administration of amonoclonal antibody as described herein. An additional therapeutic agentmay include, for example, chemotherapy, radiation therapy, etc.Additional therapeutic agents may be administered separately or as partof a mixture or cocktail. In some aspects of the present disclosure, theadministration of an antibody may allow for the effectiveness of a lowerdosage of other therapeutic modalities when compared to theadministration of the other therapeutic modalities alone, providingrelief from the toxicity observed with the administration of higherdoses of the other modalities.

In some aspects of the methods of the present disclosure, theadministration of a composition as described herein and the at least oneadditional therapeutic agent demonstrate therapeutic synergy. In someaspects of the methods of the present disclosure, a measurement ofresponse to treatment observed after administering both an antibody asdescribed herein and the additional therapeutic agent is improved overthe same measurement of response to treatment observed afteradministering either the antibody or the additional therapeutic agentalone.

EXEMPLARY EMBODIMENTS Embodiment 1

A monoclonal antibody that binds to VSIG-4.

Embodiment 2

A monoclonal antibody produced by at least one of the following clones:

Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 3

A monoclonal antibody, wherein the monoclonal antibody comprises

at least one of

-   -   a heavy chain variable region of a monoclonal antibody produced        by at least one of the following clones:    -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186);

and

-   -   a light chain variable region of a monoclonal antibody produced        by at least one of the following clones:    -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 4

A monoclonal antibody, wherein the monoclonal antibody comprises:

at least one of

-   -   a heavy chain variable region comprising a complementarity        determining region (CDR) of the heavy chain of a monoclonal        antibody produced by at least one of the following clones:    -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186);        and    -   a light chain variable region comprising a complementarity        determining region (CDR) of the light chain of a monoclonal        antibody produced by at least one of the following clones:    -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 5

The monoclonal antibody of Embodiment 4, wherein the monoclonal antibodycomprises each of the CDRs of the heavy chain of a monoclonal antibodyproduced by one of the following clones:

Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 6

The monoclonal antibody of either of Embodiments 4 or 6, wherein themonoclonal antibody comprises each of the CDRs of the light chain of amonoclonal antibody produced by one of the following clones:

Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);

Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);

Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);

Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);

Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);

Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);

Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);

Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);

Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);

Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);

Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and

Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 7

A monoclonal antibody, wherein the monoclonal antibody comprises anamino acid sequence that is

at least 80% identical to the amino acid sequence of at least onecomplementarity determining region (CDR) of the heavy chain variableregion of a monoclonal antibody produced by at least one of thefollowing clones:

-   -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186);        or

at least 80% identical to the amino acid sequence of at least onecomplementarity determining region (CDR) of the light chain variableregion of a monoclonal antibody produced by at least one of thefollowing clones:

-   -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186);        or both.

Embodiment 8

The monoclonal antibody of Embodiment 7, wherein the monoclonal antibodycomprises an amino acid sequence that is

at least 80% identical to the amino acid sequence of each of thecomplementarity determining regions (CDRs) of the heavy chain variableregion of a monoclonal antibody produced by at least one of thefollowing clones:

-   -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186);        and

at least 80% identical to the amino acid sequence of each of thecomplementarity determining regions (CDRs) of the light chain variableregion of a monoclonal antibody produced by at least one of thefollowing clones:

-   -   Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187);    -   Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188);    -   Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189);    -   Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178);    -   Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179);    -   Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180);    -   Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181);    -   Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182);    -   Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183);    -   Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184);    -   Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and    -   Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).

Embodiment 9

The monoclonal antibody of any one of Embodiments 2 to 8, wherein theantibody binds to VSIG-4.

Embodiment 10

The monoclonal antibody of any one of Embodiments 1 to 9, wherein themonoclonal antibody is coupled directly or indirectly to a detectablemarker.

Embodiment 11

The monoclonal antibody of any one of Embodiments 1 to 10, wherein themonoclonal antibody comprises an IgG antibody.

Embodiment 12

The monoclonal antibody of any one of Embodiments 1 to 11, wherein themonoclonal antibody abrogates the binding of VSIG-4 to a VSIG-4 ligand.

Embodiment 13

The monoclonal antibody of any one of Embodiments 1 to 12, wherein theVSIG-4 ligand comprises SIGLEC-7.

Embodiment 14

The monoclonal antibody of Embodiment 12 or 13, wherein at least one ofVSIG-4 and the VSIG-4 ligand are on a cell surface.

Embodiment 15

The monoclonal antibody of any one of Embodiments 12 to 14, whereinVSIG-4 is on the surface of an M2c macrophage.

Embodiment 16

The monoclonal antibody of any one of Embodiments 12 to 15, wherein themonoclonal antibody abrogates a cell-cell interaction.

Embodiment 17

The monoclonal antibody of any one of Embodiments 1 to 16, wherein themonoclonal antibody binds to an extracellular domain of VSIG-4.

Embodiment 18

The monoclonal antibody of any one of Embodiments 1 to 17, wherein themonoclonal antibody binds to a VSIG-4 polypeptide.

Embodiment 19

The monoclonal antibody of any one of Embodiments 1 to 18, wherein themonoclonal antibody binds to glycosylated and unglycosylated VSIG-4.

Embodiment 20

The monoclonal antibody of any one of Embodiments 1 to 19, wherein themonoclonal antibody comprises an antigen-binding fragment comprising atleast one of a Fab fragment, a Fab′ fragment, a F(ab)₂ fragment, and aFv fragment.

Embodiment 21

A composition comprising the monoclonal antibody of any one ofEmbodiments 1 to 20.

Embodiment 22

A kit comprising the monoclonal antibody of any one of Embodiments 1 to20.

Embodiment 23

The monoclonal antibody of any one of Embodiments 1 to 20 for use in amethod of treating a mammalian cancer.

Embodiment 24

The monoclonal antibody of any one of Embodiments 1 to 20 for use in amethod of treating an autoimmune disease.

Embodiment 25

A method of treating a mammalian cancer comprising exposing a mammalcomprising a mammalian cancer cell to the monoclonal antibody of any oneof Embodiments 1 to 20.

Embodiment 26

The method of Embodiment 25 wherein VSIG-4 expression is amplified in apatient sample comprising the mammalian cancer cell.

Embodiment 27

The method of Embodiment 25 wherein VSIG-4 expression is amplified in amacrophage of the patient sample comprising the mammalian cancer cell.

Embodiment 28

The method any one of Embodiments 25 to 27 wherein the mammalian cancercell comprises a lung cancer cell or a glioma.

Embodiment 29

The method of any one of Embodiments 25 to 28 wherein the mammaliancancer cell is also exposed to chemotherapy or radiation therapy.

Embodiment 30

A method of detecting cancer in a mammal comprising exposing a mammaliancancer cell to the monoclonal antibody of any one of Embodiments 1 to20.

Embodiment 31

A method of treating an autoimmune disease in a mammal comprisingexposing a cell of the mammal to the monoclonal antibody of any one ofEmbodiments 1 to 20.

Embodiment 32

The method of Embodiment 31 wherein VSIG-4 expression is amplified in apatient sample comprising the mammalian cell.

Embodiment 33

The method of Embodiment 32 wherein VSIG-4 expression is amplified in amacrophage of the patient sample comprising the mammalian cell.

Embodiment 34

The method of any one of Embodiments 31 to 33, wherein the autoimmunedisease comprises autoimmune type 1 diabetes mellitus, rheumatoidarthritis, psoriasis, or lupus.

Embodiment 35

The monoclonal antibody of any one of Embodiments 1 to 20 for use in amethod of treating an autoimmune disease.

Exemplary Hybridoma Cell Line Embodiments

Embodiment 1

A hybridoma cell line Ms×hVSIG4 528902.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124187.

Embodiment 2

A hybridoma cell line Ms×hVSIG4 528903.111 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124188.

Embodiment 3

A hybridoma cell line Ms×hVSIG4 528905.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124189.

Embodiment 4

A hybridoma cell line Ms×hVSIG4 528906.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124178.

Embodiment 5

A hybridoma cell line Ms×hVSIG4 528908.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124179.

Embodiment 6

A hybridoma cell line Ms×hVSIG4 528910.111 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124180.

Embodiment 7

A hybridoma cell line Ms×hVSIG4 528912.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124181.

Embodiment 8

A hybridoma cell line Ms×hVSIG4 528922.111 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124182.

Embodiment 9

A hybridoma cell line Ms×hVSIG4 528927.111 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124183.

Embodiment 10

A hybridoma cell line Rt×hVSIG4 489509.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124184.

Embodiment 11

A hybridoma cell line Rt×hVSIG4 489517.111 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124185.

Embodiment 12

A hybridoma cell line Rt×hVSIG4 489518.11 deposited with the AmericanType Culture Collection (ATCC) as ATCC accession number PTA-124186.

Exemplary Monoclonal Antibody Produced by Hybridoma Cell LineEmbodiments

Embodiment 1

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528902.11 deposited as ATCC accession number PTA-124187.

Embodiment 2

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528903.111 deposited as ATCC accession number PTA-124188.

Embodiment 3

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528905.11 deposited as ATCC accession number PTA-124189.

Embodiment 4

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528906.11 deposited as ATCC accession number PTA-124178.

Embodiment 5

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528908.11 deposited as ATCC accession number PTA-124179.

Embodiment 6

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528910.111 deposited as ATCC accession number PTA-124180.

Embodiment 7

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528912.11 deposited as ATCC accession number PTA-124181.

Embodiment 8

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528922.111 deposited as ATCC accession number PTA-124182.

Embodiment 9

A monoclonal antibody produced by the hybridoma cell line Ms×hVSIG4528927.111 deposited as ATCC accession number PTA-124183.

Embodiment 10

A monoclonal antibody produced by the hybridoma cell line Rt×hVSIG4489509.11 deposited as ATCC accession number PTA-124184.

Embodiment 11

A monoclonal antibody produced by the hybridoma cell line Rt×hVSIG4489517.111 deposited as ATCC accession number PTA-124185.

Embodiment 12

A monoclonal antibody produced by the hybridoma cell line Rt×hVSIG4489518.11 deposited as ATCC accession number PTA-124186.

Embodiment 13

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528902.11 deposited as ATCC accession number PTA-124187.

Embodiment 14

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528903.111 deposited as ATCC accession number PTA-124188.

Embodiment 15

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528905.11 deposited as ATCC accession number PTA-124189.

Embodiment 16

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528906.11 deposited as ATCC accession number PTA-124178.

Embodiment 17

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528908.11 deposited as ATCC accession number PTA-124179.

Embodiment 18

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528910.111 deposited as ATCC accession number PTA-124180.

Embodiment 19

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528912.11 deposited as ATCC accession number PTA-124181.

Embodiment 20

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528922.111 deposited as ATCC accession number PTA-124182.

Embodiment 21

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineMs×hVSIG4 528927.111 deposited as ATCC accession number PTA-124183.

Embodiment 22

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineRt×hVSIG4 489509.11 deposited as ATCC accession number PTA-124184.

Embodiment 23

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineRt×hVSIG4 489517.111 deposited as ATCC accession number PTA-124185.

Embodiment 24

A monoclonal antibody comprising the complementarity-determining regions(CDRs) of the monoclonal antibody produced by the hybridoma cell lineRt×hVSIG4 489518.11 deposited as ATCC accession number PTA-124186.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES

Unless otherwise indicated, all incubations in the following exampleswere at room temperature and all reagents, starting materials, andsolvents used were purchased from commercial suppliers (such asSigma-Aldrich, St. Louis, Mo.) and were used without furtherpurification unless otherwise indicated.

Example 1—Characterization of VSIG4-Siglec7 Interaction

Human Siglec7 (hSiglec7) (Uniprot number: Q9Y286) and human VSIG4(hVSIG4) (Uniprot number: Q9Y279; Gene ID 11326) were expressedrecombinantly in HEK293, NSO, or CHO cells to provide recombinant humanSiglec7 (rhSiglec7) and recombinant human VSIG4 (rhVSIG4). Proteins weretagged with a variety of N- or C-terminal tags, as indicated, such asHis, Fc or GFP, to enable purification and analysis in the variousassays.

Interaction of rhVSIG4 and Biotinylated rhSiglec7.

Soluble rhVSIG4 was coated on a standard ELISA plate and, after removingexcess protein and blocking additional binding sites on the plate withBSA, incubated with soluble rhSIGLEC 7. Biotinylated rhSIGLEC-7 wasdetected using Streptavidin-horseradish peroxidase (HRP). Afterbackground correction, an absorbance of 2.3 was detected at 452 nm in astandard plate reader.

HEK/eGFP Transfectant and Fc Protein Flow Screen.

eGFP-transfected HEK293 cells expressing either human VSIG-4 or humanSiglec-7 were harvested, washed with RDFII staining buffer (0.5% (w/v)BSA, 10 mM HEPES, 100 mM Sodium Chloride, 0.01% Sodium Azide, pH 7.2),counted, aliquoted at 100,000 cells/sample, and incubated with varyingamounts of recombinant human Siglec-7 or VSIG-4 proteins containing ahuman IgG Fc region (rhSiglec-7/Fc or rhVSIG-4/Fc). Proteinconcentrations tested were between 500 ng/mL and 6 μg/mL. Thetransfectants and Fc proteins were incubated for 30 minutes at roomtemperature, then anti-human IgG Fc-APC detection antibody (FAB110A, R&DSystems, Minneapolis, Minn.) was added for another 20 minutes. The cellswere then washed with RDFII staining buffer and analyzed on either aNovocyte flow cytometer (ACEA Biosciences, Inc., San Diego, Calif.) or aLSRII FORTESSA flow cytometer (BD Biosciences, San Jose, Calif.).

Example 2—Identification of Anti-VSIG4 Antibodies and VSIG4-Siglec7Blocking Antibodies

Using panels of monoclonal antibodies made against the human or mouseVSIG4 protein (Uniprot number: Q9Y279 or F6TUL9, respectively),antibodies that bind to VSIG-4 were identified by direct ELISA and FlowCytometry. Clones producing the VSIG-4-binding antibodies are listed inTable 1.

The ability of these anti-VSIG-4 antibodies to abrogate binding ofVSIG-4 to the Siglec-7 protein was also determined, as further describedbelow.

VSIG4-Siglec7 Blocking Assay.

Human VSIG-4 HEK/eGFP transfectants were harvested, washed with RDFIIstaining buffer, counted, aliquoted at a concentration of 100,000cells/sample, and incubated with 2.5 μg/mL anti-VSIG-4 antibody for 30minutes at room temperature. The cells were washed with RDFII stainingbuffer, and 4 μg/mL hSiglec-7/Fc protein was added to each tube for 30minutes. After this incubation, anti-Fc APC detection antibody (FAB110A)was added for another 20 minutes. The cells were then washed with RDFIIstaining buffer, and analyzed on either an Aceabio Novocyte flowcytometer or a LSRII Fortessa flow cytometer (BD Biosciences, San Jose,Calif.). Exemplary results are shown in FIG. 5; additional results areshown in Table 1.

TABLE 1 Anti-VSIG-4 Monoclonal Antibodies and their characteristics HostAntibody species Type Applications Kd Blocking Clone Ms x hVSIG4528902.11 mouse IgG3 WB (neg), FC ND N Clone Ms x hVSIG4 528903.111mouse IgG2A WB, FC  7.7 nM Y Clone Ms x hVSIG4 528905.11 mouse IgG2B WB,FC ND N Clone Ms x hVSIG4 528906.11 mouse IgG2A FC  4.6 nM N Clone Ms xhVSIG4 528908.11 mouse IgG2A WB, FC  6.3 nM Y Clone Ms x hVSIG4528910.111 mouse IgG3 WB, FC ND N Clone Ms x hVSIG4 528912.11 mouseIgG2A FC 60.6 nM Y Clone Ms x hVSIG4 528922.111 mouse IgG2A WB (neg), FCND N Clone Ms x hVSIG4 528927.111 mouse IgG2B WB, FC ND N Clone Rt xhVSIG4 489509.11 Rat IgG2A FC   1 nM Y Clone Rt x hVSIG4 489517.111 RatIgG2A FC  2.1 nM Y Clone Rt x hVSIG4 489518.11 Rat IgG2A FC  0.3 nM Y ND(not determined, no binding observed in SPR assay); FC (Flow Cytometry),WB (Western Blot)

Example 3—Characterization of VSIG4-Siglec7 Blocking Antibodies

Generation of Polarized M2c Cells.

PBMCs were isolated using a SEPMATE/Ficoll gradient isolation protocolaccording to standard protocols. CD14⁺ cells were then purified using apositive selection kit for CD14 (Catalog # MAGH105, R&D Systems,Minneapolis, Minn.). One million CD14 positive cells/mL (total volume of4 mL) were placed in complete media (RPMI culture media supplementedwith 10% Fetal Bovine Serum (ThermoFisher Scientific, Waltham, Mass.),1.0 mM Sodium Pyruvate (Invitrogen, Carlsbad, Calif.), 50 mM2-mercaptoethanol (Invitrogen), 1× Glutamax (Life Technologies, GrandIsland, N.Y.), 1× Penicillin/Streptomycin (Life Technologies, GrandIsland, N.Y.), and 1× Non-Essential Amino Acids (Life Technologies,Grand Island, N.Y.)) supplemented with 50 ng/mL hM-CSF (Catalog #216-MCR&D Systems, Minneapolis, Minn.) for 4 days. On day 5, cells weretransferred into fresh media to allow cells to polarize to M2cMacrophages for an additional 3 days, the media additionally containingeither 30 ng/mL Dexamethasone (Catalog #1126, Tocris Biosciences,Minneapolis, Minn.) or 20 ng/mL rhIL-10 (Catalog #217-IL, R&D Systems,Minneapolis, Minn.).

Flow Cytometric Detection of VSIG-4 Expression in Polarized M2c Cells.

Dexamethasone polarized M2c cells were harvested on day 7, washed twotimes with 1×PBS, and incubated with Zombie Violet (BioLegend, SanDiego, Calif.) at 1 μL per 100 μL cells for 30 minutes at roomtemperature to allow for dead cell exclusion. Cells were then washed 2times with RDFII staining buffer, blocked with human IgG and goatanti-human IgG (Catalog #1597, R&D Systems, Minneapolis, Minn.) for 10minutes at 4° C. Cells were then surface stained for 30 minutes at 4° C.with mouse anti-human VSIG4 Alexa Fluor 647, anti-CD14 FITC, andanti-Mer PE (Catalog numbers, FAB982F, FAB8912P, R&D Systems,Minneapolis, Minn.). The cells were washed with RDFII staining bufferone last time and analyzed on a LSRII Fortessa flow cytometer (BDBiosciences, San Jose, Calif.). Results are shown in FIG. 6.

Detection of Siglec-7/VSIG-4 Interaction in Primary M2c Cells Polarizedwith 20 ng/mL IL-10.

Polarized M2c cells were incubated with varying amounts of hSiglec-7 Fcprotein (1 μg/mL to 10 μg/mL) for 30 minutes at room temperature. Afterthis incubation, an anti-Fc PE detection antibody (FAB110P, R&D Systems,Minneapolis, Minn.) was added for an additional 20 minutes. Followingthe incubation, the cells were washed with RDFII staining buffer, andco-stained with anti-VSIG4 Alexa Fluor 647 and anti-CD14 FITC antibodiesfor 30 minutes. Following the incubation with these co-stains, the cellswere washed again with RDFII staining buffer and analyzed on a LSRIIFortessa flow cytometer (BD Biosciences, San Jose, Calif.). Results areshown in FIG. 7.

Antibody Pairing and Co-Inhibition Studies for Antigen Recognition.

Antibody pairs were identified by ELISA using a biotinylated antibodydetection method and Streptavin-HRP as the detection modality. Resultsare shown in Table 2. Based on the ability to identify pairs ofcapture/detection antibodies for VSIG4, the clones are recognizingvarious epitopes within the protein.

TABLE 2 Antibody pairs for VSIG-4 Capture Antibody Clone DetectionAntibody Clone Rt × hVSIG4 489509.11 Ms × hVSIG4 528903.111 Ms × hVSIG4528906.11 Ms × hVSIG4 528908.11 Ms × hVSIG4 528912.11 Rt × hVSIG4489517.111 Rt × hVSIG4 489518.11 Rt × hVSIG4 489517.111 Rt × hVSIG4489509.11 Rt × hVSIG4 489518.11 Rt × hVSIG4 489509.11 Ms × hVSIG4528903.111 Rt × hVSIG4 489509.11 Ms × hVSIG4 528906.11 Rt × hVSIG4489509.11 Ms × hVSIG4 528908.11 Rt × hVSIG4 489509.11

Example 4—Glycosylation of VSIG4 and Characterization of Anti-VSIG4Antibody Binding to Glycosylated VSIG4

To determine whether Siglec7 binds to glycosylated VSIG4 via apost-translationally modified glycan moiety, rhVSIG4 was expressed in E.coli, which lack the glycosyltransferase machinery and producerecombinant proteins devoid of any glycan modifications. VSIG4 producedin E. coli lost the ability to bind Siglec7 despite retaining its knownfunction of binding to complement component iC3b, suggesting thatSiglec7 binds to the glycosylated form of VSIG4.

Enzymatic deglycosylation of VSIG4 using a standard deglycosylationenzyme kit, which contains typical sialidase enzymes that cleave α(2,3)-and α(2,6)-linked N-acetylneuraminic acid (NANA, or sialic acids),however, showed binding between VSIG4 and Siglec7 was retained. Byincorporating a specific sialidase enzyme that additionally cleaves therelatively rare α(2,8)-linked NANA in addition to α(2,3)- andα(2,6)-linked NANA, a ˜10 kD shift in the molecular weight of rhVSIG4,from ˜42 kD to the predicted ˜32 kD molecular weight, was observed.Moreover, treatment with the expanded deglycosylation enzymekitabrogated the binding interaction between VSIG4 and Siglec7.

These results indicate that VSIG4 is likely heavily modified bypolysialic acid chains via α(2,8)-linked N-acetylneuraminic acid, arelatively rare glycan linkage. The most notable protein containingα(2,8)-linked polysialic acid is CD56/NCAM. (Schnaar et al.Physiological Reviews 2014, 94(2):461-518.) An interaction betweenSiglec7 and CD56/NCAM was confirmed using Biacore, and the interactionaffinity between Siglec7-VSIG4 is roughly equivalent to the interactionaffinity between Siglec7-CD56 NCAM.

To confirm that an antibody derived against VSIG4 using themammalian-derived and fully glycosylated rhVSIG4 binds to the VSIG4polypeptide and not to the glycan-modification on VSIG4, antibodybinding was tested against the fully glycosylated rhVSIG4, thedeglycosylated VSIG4, E. coli-derived VSIG4, and CD56/NCAM. Strongbinding of the blocking antibody to all VSIG4 species regardless ofglycosylation state but not to CD56/NCAM was observed.

Collectively, these results suggest that VSIG4 is heavily modified by arare glycan linkage, a α(2,8)-linked polysialic acid, and that thismodification mediates the interaction between VSIG4 and Siglec7.Furthermore, these results indicate that the blocking antibodies bindspecifically to the VSIG4 polypeptide and not to the glycan coat ofVSIG4.

Purified recombinant human VSIG4 ectodomain (rhVSIG4) expressed in NS0cells was subjected to deglycosylation with an enzyme cocktailcontaining recombinant C.p Neuraminidase (R&D Systems catalog #5080-NM),recombinant F.m. PNGase F (R&D Systems catalog #9109-GH), recombinantE.f. O-glycosidase (R&D Systems catalog #8886-GH), and recombinant S.pB4Galactosidase (R&D Systems catalog #5549-GH), and supplemented withrecombinant M.v. Neuraminidase (R&D Systems catalog #5084-NM). PurifiedrhVSIG4, both fully glysocylated and deglycosylated, were separated bySDS-PAGE and visualized by silver stain. Results are shown in FIG. 9A.The predicted molecular weight of rhVSIG4 ectodomain in the absence ofpost-translational modifications is 32 kD, and the observed molecularweight of the fully glycosylated rhVSIG4 ECD is ˜41 kD. rhVSIG4 derivedfrom the NS0 mammalian cell line is, therefore, believed to be heavilyglycosylated, and the deglycosylation enzyme mix removes nearly allsugar molecules (with M.v. neuraminidase cleaving the 2-8 linked sialicacids).

Recombinant human Siglec7-Fc (rhSiglec7-Fc) was captured on a BiacoreCM5 chip modified with Protein A/G/L at ˜700 RU capture density andtested for binding with fully glycosylated rhVSIG4, deglycosylatedVSIG4, and CD56/NCAM at concentrations ranging between 0.5 nM and 500nM. As shown in FIG. 9B-FIG. 9D, Siglec7 interacted with rhVSIG4 but notwith the deglycosylated VSIG4. Additionally, Siglec7 did not interactwith VSIG4 expressed in E. coli. CD56/NCAM is heavily glycosylated witha sugar profile similar to VSIG4, and the interaction between Siglec7and CD56/NCAM further suggests the interaction between Siglec7 and VSIG4is mediated by a unique glycosylation profile.

Anti-VSIG-4 antibody binding to a VSIG4 polypeptide regardless ofglycosylation state was tested using Biacore. Blocking antibody(Rt×hVSIG4 489517.111, Ms×hVSIG4 528906.11) was captured on a ProteinA/G/L chip at ˜350 RU and tested for binding to rhVSIG4, mammalian NS0cell derived rhVSIG4, mammalian derived and fully deglycosylated, VSIG4,E. coli derived and free of glycosylation, and CD56/NCAM, a heavilyglycosylated protein. All analyte proteins, rhVSIG4 and CD56/NCAM weretested at concentrations ranging between 25 pM and 250 nM. Binding toVSIG4 regardless of VSIG4's glycosylation state and not to CD56/NCAM, aheavily glycosylated molecule with a similar glycoprofile as VSIG4,demonstrates the specificity of an antibody to the VSIG4 amino acidsequence and indicates the antibody is not anti-glycosylation specific.Results are shown in FIG. 10-FIG. 13.

Example 5—Further Characterization of the VSIG4-Siglec7 Interaction andVSIG4-Siglec7 Blocking Antibodies

HEK293 cells were transfected with recombinant human VSIG4-eGFP orrecombinant human VSIG3-eGFP. The cells were then incubated with a noprotein (negative control) or recombinant human Siglec-7-Fc in theindicated doses. Cells were then incubated with an anti-FcAllophycocyanin (APC). As shown in FIG. 14, recombinant human Siglec-7binds specifically to HEK293 cells transfected with recombinant humanVSIG4 fused to eGFP in a dose-dependent manner, but recombinant humanSiglec-7 does not bind to HEK293 cells type transfected with recombinanthuman VSIG3-eGFP.

Adherent M2c macrophages were polarized with Dexamethasone as describedin Example 3. M2c macrophages were incubated with 10 μg/mL, 40 μg/mL or200 μg/mL anti-human VSIG-4 antibodies to test the antibody's capacityto block the interaction with rhSiglec-7. rhSiglec-7 was tested atconcentrations of at 100 μg/mL, 25 μg/mL, or 5 μg/mL. Ms×hVSIG4528906.11 blocks the interaction of naturally expressed VSIG-4 on M2cmacrophages with recombinant human Siglec-7/Fc in a dose dependentmanner as shown in FIG. 15-FIG. 18.

Example 6—Direct ELISA Testing of Anti-VSIG-4 Antibodies

Plates were coated with a titration series of recombinant human VSIG4protein ranging from 400 ng/ml to 1.56 ng/ml in a 4-fold dilutionseries. Anti-VSIG-4 antibodies were added at 1 mg/ml and incubated for 1hour followed by goat anti-mouse or a goat anti-rat HRP-conjugatedsecondary antibodies (to match the primary clones) at a 1:10,000dilution. The HRP activity was detected by addition of TMB and stopsolution addition after 10 minutes. Results were evaluated by absorbanceat 540 nm. Representative data are shown in FIG. 19 and are summarizedin Table 3.

TABLE 3 Representative ELISA results VSIG4 VSIG Clone number from NSOfrom E. coli Ms × hVSIG4 528902.11 neg weak Ms × hVSIG4 528903.111 pospos Ms × hVSIG4 528905.11 pos neg Ms × hVSIG4 528906.11 pos pos Ms ×hVSIG4 528908.11 pos pos Ms × hVSIG4 528910.111 pos neg Ms × hVSIG4528912.11 pos pos Ms × hVSIG4 528922.111 neg pos Ms × hVSIG4 528927.111pos pos Rt × hVSIG4 489509.11 pos pos Rt × hVSIG4 489517.111 pos pos Rt× hVSIG4 489518.11 pos pos

As shown in Table 3, most antibodies tested detected both glycosylatedhuman VSIG4 expressed in NS0 as well as non-glycosylated recombinanthuman VSIG4 protein expressed in E. coli, indicating that the antibodiesdetect the amino acid component of the protein and not secondarymodifications.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, for example, GenBank andRefSeq, and amino acid sequence submissions in, for example, SwissProt,PIR, PRF, PDB, and translations from annotated coding regions in GenBankand RefSeq) cited herein are incorporated by reference. In the eventthat any inconsistency exists between the disclosure of the presentapplication and the disclosure(s) of any document incorporated herein byreference, the disclosure of the present application shall govern. Theforegoing detailed description and examples have been given for clarityof understanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

What is claimed is:
 1. A monoclonal antibody produced by at least one of the following clones: Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187); Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188); Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189); Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178); Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179); Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180); Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181); Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182); Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183); Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184); Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); and Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).
 2. The monoclonal antibody of claim 1, wherein the antibody binds to VSIG-4.
 3. A monoclonal antibody, wherein the monoclonal antibody comprises: a heavy chain variable region comprising the three complementarity determining regions (CDRs) of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528902.11 (ATCC Accession No. PTA-124187); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528903.111 (ATCC Accession No. PTA-124188); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528905.11 (ATCC Accession No. PTA-124189); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528906.11 (ATCC Accession No. PTA-124178); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528908.11 (ATCC Accession No. PTA-124179); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528910.111 (ATCC Accession No. PTA-124180); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528912.11 (ATCC Accession No. PTA-124181); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528922.111 (ATCC Accession No. PTA-124182); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Ms×hVSIG4 528927.111 (ATCC Accession No. PTA-124183); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Rt×hVSIG4 489509.11 (ATCC Accession No. PTA-124184); a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Rt×hVSIG4 489517.111 (ATCC Accession No. PTA-124185); or a heavy chain variable region comprising the three CDRs of the heavy chain of a monoclonal antibody produced by Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186), and a light chain variable region comprising the three CDRs of the light chain of a monoclonal antibody produced by Rt×hVSIG4 489518.11 (ATCC Accession No. PTA-124186).
 4. The monoclonal antibody of claim 3, wherein the antibody binds to VSIG-4.
 5. The monoclonal antibody of claim 4, wherein the monoclonal antibody abrogates the binding of VSIG-4 to SIGLEC-7.
 6. The monoclonal antibody of claim 5, wherein VSIG-4 or SIGLEC-7 or both are on a cell surface.
 7. The monoclonal antibody of claim 5, wherein VSIG-4 is on the surface of an M2c macrophage.
 8. The monoclonal antibody of claim 5, wherein the monoclonal antibody abrogates a cell-cell interaction.
 9. The monoclonal antibody of claim 4, wherein the monoclonal antibody binds to an extracellular domain of VSIG-4.
 10. The monoclonal antibody of claim 4, wherein the monoclonal antibody binds to a VSIG-4 polypeptide.
 11. The monoclonal antibody of claim 4, wherein the monoclonal antibody binds to glycosylated and unglycosylated VSIG-4.
 12. The monoclonal antibody of claim 4, wherein the monoclonal antibody is coupled directly or indirectly to a detectable marker.
 13. The monoclonal antibody of claim 4, wherein the monoclonal antibody comprises an IgG antibody.
 14. The monoclonal antibody of claim 4, wherein the monoclonal antibody comprises an antigen-binding fragment comprising at least one of a Fab fragment, a Fab′ fragment, a F(ab)₂ fragment, and a FIT fragment.
 15. A composition comprising the monoclonal antibody of claim
 4. 16. A kit comprising the monoclonal antibody of claim
 4. 17. The monoclonal antibody of claim 4, wherein VSIG-4 is on the surface of an M2c macrophage.
 18. The monoclonal antibody of claim 4, wherein the monoclonal antibody abrogates a cell-cell interaction. 